Starch is a mixture of two components. One component is a linear amylose in which glucose are linked by α-1,4 linkage and the other component is an amylopectin which has a branched structure of glucose via α-1,6 linkage. These components are synthesized by the actions of various enzymes and in the case of cereals, they are accumulated in the endosperm of seeds. An amylose has been found to be mainly synthesized by Granule Bound Starch Synthase encoded by Granule Bound Starch Synthase gene. On the other hand, an amylopectin is synthesized by the actions of multiple enzymes. The enzymes include (soluble) Starch Synthase I, (soluble) Starch Synthase II, (soluble) Starch Synthase III, branching enzyme, debranching enzyme and the like.
Starch is also accumulated in the form of a grain highly crystallized in a plant. By adding water to this and heating it, the starch grain gradually swells and then the crystal structure is broken in one breath at a certain temperature (gelatinization peak temperature) to be pasty (gelatinized). Subsequently, on cooling the gelatinized starch, it is gradually increased in its viscosity to be gelled (retrogradated). It has been known that such a property and the ratio of amylose and amylopectin are greatly different depending on the plant species.
Starch is a reserve substance in a plant, as well as an important energy source for animals. In taking starch, not only cereals containing it are utilized as processed foodstuffs, but also it is used as an additive such as a thickener, water retention agent and gel-forming agent by making use of the above property. On the other hand, starch has been also used as a raw material of glue and film in industry. In addition, there is a lot of demands for processed starch which is chemically or physically modified. Starch occupies the majority of the quantity of the organ (seed or tuber) where starch is accumulated. The changes in the starch property highly affect the eating quality or processability of the above products making use of the property, so a demand for the development of starch having diverse properties is large.
The property of starch as described above is greatly different depending on the plant species. However, the diversity of starch in the same plant species owes much to the change of physical property due to the difference of amylose content. For example, in a wheat, the amylose content is about 30% in the usual type of starch, but a lower amylose line whose amylose content is about 20% has been known. The wheat starch of the lower amylose line is considered to be superior to one of the usual type in use as flour for noodle such as Japanese wheat noodle, it has been also commercially cultivated widely. Moreover, in rice and corn, the type in which waxy-type starch whose amylose content is extremely low is accumulated has been known. In a wheat, however, the waxy-type starch has been bred for the first time by Nakamura et al. (JP-A-6-125669 (the term “JP-A” as used herein means an “unexamined published Japanese patent application”), and it has been known to have unique processability and food quality compared to those of usual type. The amylose content has been discussed as one of features representing the characteristics of starch, but in the case of the same plant species, the diversity except for the amylose content is low. Therefore, the diversity of starch from wheat or wheat flour containing it become low, only leading to uniform commodities, so the market is felt to have been already matured. For this, if a wheat which accumulates a starch having a new property can be developed, it allows the development of an improved product providing features different from conventional ones or a new application. The development of such a wheat, therefore, has been desired.
As one example of producing a wheat having a new property, Yamamori et al. reports that they have developed a wheat line in which Wheat Starch Synthase II, one of enzymes which synthesize amylopectin branched chain, is deleted (see Yamamori et al., Theor. Appl. Genet (2000) 101: 21-29). Although it has also been reported that such wheat accumulates high content of amylose, study on other properties has been hardly proceeded, and it has not arrived a practical application.
In a wheat, an amylose is synthesized by Granule Bound Starch Synthase encoded by Granule Bound Starch Synthase gene. In a wheat chromosome which is an allohexaploid, three genomes A, B, D that are homologous chromosomes. Usually, three Granule Bound Starch Synthase genes would exist, and Granule Bound Starch Synthase would be expressed by these genes (Granule Bound Starch Synthase A1, Granule Bound Starch Synthase B1 and Granule Bound Starch Synthase D1). However, there exist the type of chromosomes in which the protein is not expressed due to the mutation produced on the genome DNA, result in 8 combinations of the expression, including wild types. It is known to be found a significant difference in the amylose content according to this pattern. The wheat line in which 1 or 2 proteins are deleted is a lower amylose line, and the type in which all of three proteins are deleted results in glutinous wheat. As a simple method for distinguishing these deleted patterns, a method for directly analyzing proteins expressed in endosperm and a method for investigating based on genome DNA sequences are established (see, for example, JP-A-6-125669 and Nakamura et al., (2002) Genome 45: 1150-1156).
On the other hand, Wheat Starch Synthase II Protein has been known as one of enzymes which are involved in the synthesis of branched chains of amylopection. Each Protein of Wheat Starch Synthase II is encoded by each of three Wheat Starch Synthase II genes located on 7A, 7B and 7D chromosome respectively (Wheat Starch Synthase II-A1, Wheat Starch Synthase II-B1 and Wheat Starch Synthase II-D1). As for Wheat Starch Synthase II Protein also, a method for distinguish deleted patterns has been already developed by present inventors. Although expression patterns can be also divided into 8 combinations as for Wheat Starch Synthase II Protein using this method, the sufficient research on starch property in each pattern has not been made.